The present invention relates to fluorescent proteins, in particular green fluorescent proteins (GFPs), with increased activity in cells, and thus increased signal strength. A further aspect of the present invention relates to the use of peptides for increasing the expression and/or stability of a protein in a cell.
Because of its easily detectable green fluorescence, green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been widely used to study gene expression and protein localization. GFP fluorescence does not require a substrate or cofactor; hence, it is possible to use this reporter in a wide variety of applications and cells.
The green fluorescent protein (GFP) is a protein composed of 238 amino acids (26.9 kDa), which exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from A. victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum.
GFP has a typical beta barrel structure, consisting of one β-sheet with alpha helices containing the chromophore running through the centre. Inward facing sidechains of the barrel induce specific cyclization reactions in the tripeptide Ser65-Tyr66-Gly67 that lead to chromophore formation. This process of post-translational modification is referred to as maturation. The hydrogen bonding network and electron stacking interactions with these sidechains influence the colour of wildtype GFP and its numerous derivatives. The tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water.
Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered (Shaner et al., 2005). The first major improvement was a single point mutation (S65T) reported in 1995. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability and a shift of the major excitation peak to 488 nm with the peak emission kept at 509 nm. This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher. A 37° C. folding efficiency (F64L) point mutant yielding enhanced GFP (EGFP) was discovered in 1995 and facilitated the use of GFPs in mammalian cells. Superfolder GFP, based on a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.
Many other mutations have been made, including colour mutants; in particular blue fluorescent protein, cyan fluorescent protein and yellow fluorescent protein derivatives. BFP derivatives contain the Y66H substitution. The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an indole rather than phenol component. The red-shifted wavelength of the YFP derivatives is accomplished by the T203Y mutation and is due to π-electron stacking interactions between the substituted tyrosine residue and the chromophore.
Semirational mutagenesis of a number of residues led to pH-sensitive mutants known as pHluorins, and later super-ecliptic pHluorins. By exploiting the rapid change in pH upon synaptic vesicle fusion, pHluorins tagged to synaptobrevin have been used to visualize synaptic activity in neurons.
Redox sensitive versions of GFP (roGFP) were engineered by introduction of cysteines into the beta barrel structure. The redox state of the cysteines determines the fluorescent properties of roGFP.